The present invention relates to methods of modulating bone growth and, remodeling, methods of treating or preventing bone diseases, methods of inducing and stimulating bone growth and peak bone mass or repair, pharmaceutical compositions for modulating bone growth, articles of manufacturing and methods of identifying bone growth modulating agents. Specifically, the present invention employs regulating the expression or activity of cannabinoid receptors in bone cells which in turn modulates bone growth and remodeling.
Naturally occurring cannabinoids may be divided into two categories, plant-derived and endogenous. Plant-derived cannabinoids are known to elicit dramatic psychobehavioral effects, exemplified by the well-known Δ9-tetrahydrocannabinol (THC), the psychotropic principle in marijuana. They are also known to have complex cardiovascular effects, a prominent component of which is hypotension [Vollmer et al., J. Pharm. Pharmacol. 26:186-198 (1974)]. Endogenous cannabinoids (endocannabinoids) are a class of lipid-like molecules that share receptor binding sites with plant-derived cannabinoids and mimic many of their neurobehavioral effects [Mechoulam et al., Adv. Exp. Bio. Med. 402:95-101 (1996)]. Two endocannabinoids have been characterized in some detail: arachidonyl ethanolamide (anandamide) [Devane et al., Science 258:1946-1949 (1992); Felder et al., Proc. Natl. Acad. Sci. USA. 90:7656-7660 (1993)] and 2-arachidonoyl glycerol (2-AG) [Mechoulam et al., Biochem. Pharmacol 50:83-90 (1995)].
Additional natural or synthetic cannabinoids are described in U.S. Pat. Nos. 4,371,720, 5,013,387, 5,081,122, 5,292,736, 5,461,034, 5,618,955, 6,166,066 and 6,531,636; International Patent applications WO 01/9773, WO 97/29079, WO 99/02499, WO 98/41519, and WO 94/12466; European Patent Nos. EP 0570920 and EP 0444451; French Patent No. FR 2735774; and Israeli Pat. Nos. IL 01/00551 and IL 99/00187; Gaoni and Mechoulam, J. Amer. Chem. Soc. 93, 217 (1971); Mechoulam et al., Science 169, 611 (1970); Edery et al., Ann. N.Y. Acad. Sci., 191, 40 (1971); Mechoulam et al., J. Amer. Chem. Soc., 94, 7930 (1972); R. Mechoulam (ed.), “Marijuana: Chemistry, Metabolism, Pharmacology, and Clinical Effects” Academic Press, 1973, New-York; Houry et al., J. Med. Chem., 17, 287 (1974); Houry et al., J. Med. Chem., 18, 951 (1975); Mechoulam et al., Chem. Reviews, 76, 75 (1976); Mechoulam et al., J. Med. Chem., 23, 1068 (1980); Srebnik et al., J. Chem. Soc., Perkin Trans. I, 2881 (1984); Mechoulam et al., Tetrahedron: Asymmetry, 1, 315 (1990); Devane et al., Science, 258, 1946 (1992); Burstein et al., J. Med. Chem., 35, 3135 (1992); Hanus et al., J. Med. Chem., 36, 3032 (1993); Mechoulam et al., Biochem. Pharmacol., 50, 83 (1995); Sheskin et al., J. Med. Chem., 40, 659 (1997); Rhee et al., J. Med. Chem. 40, 3228 (1997); and Hanus et al., PNAS, 98, 3662 (2001).
Endocannabinoids exert their effects by binding to specific receptors thereby activating neurotransmitters and hormone regulators [Piomelli et al., Trends Pharmacol. Sci. 21: 218-224 (2000); Petwee, R. G., Curr. Med. Chem. 6:635-664 (1999); and Devane et al., J. Med. Chem. 35: 2065-2069)].
To date, two types of high-affinity cannabinoid receptors have been identified by molecular cloning: (i) CB1 receptors, present mostly in brain [Devane et al., Mol. Pharmacol. 34:605-613 (1988); Matsuda et al., Nature 346:561-564 (1990)] but also in some peripheral tissues [Shire et al., J. Biol. Chem. 270:3726-3731 (1995); Ishac et al., Br. J. Pharmacol. 118:2023-2028 (1996)], and (ii) CB2 receptors, present on macrophages in the spleen [Munro et al., Nature 365:61-65 (1993)]. Other types or subtypes of cannabinoid receptors have been recently described, designated CB1-like receptors, CB2-like receptors, and non-CB1 non-CB2 receptors [Hanus et al., J. Pharmacol. Exper. Therapeutics 54: 161-202 (2002)].
The physiological roles of endogenous cannabinoids and the pathways of endocannabinoid signaling are the subject of intense investigation and have been reported to affect processes in the nervous, cardiovascular, immune, and reproductive systems [Mechoulam et al., Eur. J. Pharmacol. 359: 1-18 (1998); Axelrod and Felder, Neurochem. Res. 23: 575-581(1998); Wagner et al., J. Mol. Med. 76: 824-836 (1999); and Klein et al., Immunol. Today 19: 373-381 (1998)].
Accordingly, cannabinoids or cannabinoids receptor ligands have been used or described as useful therapeutic agents for treating a variety of medical disorders.
Thus, THC has been extensively used to prevent excessive weight loss by cancer or AIDS patients [Mechoulam et al., E. Prog. Med. Chem. 35: 199-243 (1998)].
U.S. Pat. No. 5,939,429 discloses use of agonists of CB1 receptors as well as other cannabinoid receptors to treat cardiovascular conditions, including hemorrhagic shock and in other conditions associated with excessive vasoconstriction, such as hypertension, peripheral vascular disease, cirrhosis of the liver, and certain forms of angina pectoris. In addition it teaches use of antagonists of CB1 and other cannabinoid receptors for treating hypotension which is caused by endotoxin activation of macrophages.
U.S. Pat. No. 6,166,066 discloses use of cannabinoids which are selective for the CB2 receptor as immunosuppressive agents for preventing tissue rejection in organ transplant patients and for treating autoimmune associated diseases.
U.S. application Ser. No. 09/779,109 discloses use of cannabinoids receptor modulators for treating respiratory or non-respiratory leukocyte-activation associated diseases. Exemplary non-respiratory cannabinoid receptor-mediated diseases include transplant rejection, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, lupus, graft v. host disease, T-cell mediated hypersensitivity disease, psoriasis, Hashimoto's thyroiditis, Guillain-Barre syndrome, cancer, contact dermatitis, allergic rhinitis, and ischemic or reperfusion injury.
U.S. application Ser. No. 10/032,163 discloses a method of increasing the activity of a cannabinoid agonist that binds specifically to an endogenous cannabinoid receptor, so as to protect the cells against glutamate-induced neurotoxicity.
Yet, while cannabinoids or cannabinoid receptor ligands have been suggested for use as therapeutic agents, application of cannabinoids for treating or preventing bone-related diseases has never been described nor suggested in prior art.
While reducing the present invention to practice, the inventors of the present invention surprisingly uncovered the major role of endocannabinoids in modulating bone growth and remodeling, thus indicating the potential benefits of using cannabinoids or cannabinoids receptor ligands as therapeutic agents for treating bone diseases and injuries as well as promoting bone formation or inhibiting bone resorption.
Bone is subject to constant breakdown and resynthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. This process is referred to as bone remodeling. The activities of these cells are regulated by a large number of cytokines, hormones and growth factors, many of which have now been identified and cloned.
There is a plethora of conditions which are characterized by the need to promote bone formation and/or to inhibit bone resorption. Perhaps the most obvious is the case of bone fractures, where it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in endosseous implants and facial reconstruction procedures, and of great importance in the growing field of prosthetic and therapeutic bone implants. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, osteoporosis associated with depression and hypogonadism and glucocorticoid-related osteoporosis.
On the other hand, there are conditions which are characterized by the need to inhibit bone formation or to promote bone resorption. These include certain stages of Paget's disease, blastic metastatic bone cancer, Hodgkin's lymphoma, degenerative sclerosis and osteomyelitis. Agents known to be effective in inhibition of bone growth, and in bone resorption, are the cyclooxygenase inhibitors, 1, 25 (OH)2 vitamin D3, the glucocorticoids, omeprazole, the serum protein fetuin, noggin, blockers of beta adrenergic receptors, chordin and DAN proteins and high concentrations of TGF-beta. However, all of the abovementioned agents (particularly the glucocorticoids and other hormones) are known to exert their influence on a wide variety of tissues, and as such are unsuited for pharmacological applications in bone diseases.
Various therapeutic agents and approaches to treatment of bone related diseases have been disclosed in patent publications.
U.S. Pat. No. 5,461,034 discloses osteogenic growth polypeptides identified from regenerating bone marrow, for the enhancement of bone formation and bone marrow in preparation for bone marrow transplant. U.S. Pat. No. 5,280,040 discloses antiestrogenic, oral contraceptive compounds, 3,4-diarylchromans, described as useful in the treatment of osteoporosis. U.S. Pat. No. 6,352,973 discloses a recombinant protein containing a bone morphogenic polypeptide of the TGF-beta superfamily of cytokines originally isolated from blood serum, for enhancing bone growth. U.S. Pat. No. 6,462,019 discloses inhibitors of proteasomal activity and production for inhibiting osteoclastic activity and stimulating bone growth, based on the observation that mice lacking proteasomal activity develop the condition of excess bone formation known as osteopetrosis.
International patent application No. 92/15615 discloses a protein derived from a porcine pancreas which acts to depress serum calcium levels for treatment of bone disorders that cause elevation of serum calcium levels.
International patent application No. 92/14481 discloses a composition for inducing bone growth which contains activin and bone morphogenic protein.
European Patent Application No. 504 938 discloses the use of di- or tripeptides which inhibit cysteine protease in the treatment of bone diseases.
European Patent Application No. 499 242 discloses the use of cell growth factor compositions thought to be useful in bone diseases involving bone mass reduction because they cause osteoblast proliferation.
European Patent Application No. 451 867 discloses parathyroid hormone peptide antagonists for treating dysbolism associated with calcium or phosphoric acid, such as osteoporosis.
Yet, currently no satisfactory pharmaceutical approaches to managing bone defects are available. Bone fractures are still treated exclusively using casts, braces, anchoring devices and other strictly mechanical means. Further bone deterioration associated with osteoporoses has been treated with estrogens or bisphosphonates, which may have drawbacks for some individuals.
Although the Bone Morphogenic Proteins (BMPs) are potent stimulators of bone formation in vitro and in vivo, there are disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that BMPs may have effects on many tissues in addition to bone, potentially limiting their usefulness as therapeutic agents when administered systemically. Moreover, since they are peptides, they would have to be administered by injection. These disadvantages impose severe limitations to the development of BMPs as therapeutic agents.
The fluorides, suggested also for the purpose of enhancing bone formation, have a mode of action which may be related to tyrosine phosphorylation of growth factor receptors on osteoblasts, as described, for example, Burgener et al. J Bone Min Res (1995) 10:164-171, but administration of fluorides is associated with increased bone fragility, presumably due to adverse effects on bone mineralization.
Parathyroid hormone, currently considered the leading agent for metabolic enhancement of bone formation, is inherently problematic, since it is only administered by injection.
Thus, although various approaches have been tried, such as described above, there remains a need for additions to the repertoire of agents which can be used to treat these conditions.
There is thus a widely recognized need for, and it would be highly advantageous to have novel effective bone growth modulating agents acting through normal signaling pathways, which can be used to treat these conditions. Accordingly, the present invention provides novel methods, pharmaceutical compositions and articles of manufacture for modulating bone growth and for treating or preventing bone defects based on regulating cannabinoid receptors.